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A multi-animal tracker for studying complex behaviors

View Article: PubMed Central - PubMed

ABSTRACT

Background: Animals exhibit astonishingly complex behaviors. Studying the subtle features of these behaviors requires quantitative, high-throughput, and accurate systems that can cope with the often rich perplexing data.

Results: Here, we present a Multi-Animal Tracker (MAT) that provides a user-friendly, end-to-end solution for imaging, tracking, and analyzing complex behaviors of multiple animals simultaneously. At the core of the tracker is a machine learning algorithm that provides immense flexibility to image various animals (e.g., worms, flies, zebrafish, etc.) under different experimental setups and conditions. Focusing on C. elegans worms, we demonstrate the vast advantages of using this MAT in studying complex behaviors. Beginning with chemotaxis, we show that approximately 100 animals can be tracked simultaneously, providing rich behavioral data. Interestingly, we reveal that worms’ directional changes are biased, rather than random – a strategy that significantly enhances chemotaxis performance. Next, we show that worms can integrate environmental information and that directional changes mediate the enhanced chemotaxis towards richer environments. Finally, offering high-throughput and accurate tracking, we show that the system is highly suitable for longitudinal studies of aging- and proteotoxicity-associated locomotion deficits, enabling large-scale drug and genetic screens.

Conclusions: Together, our tracker provides a powerful and simple system to study complex behaviors in a quantitative, high-throughput, and accurate manner.

Electronic supplementary material: The online version of this article (doi:10.1186/s12915-017-0363-9) contains supplementary material, which is available to authorized users.

No MeSH data available.


The Multi-Animal Tracker (MAT) is compatible with accurate high-throughput measurements of aging- and neurodegenerative-associated locomotion decline. a Synchronized CF512 worms were grown to 1, 4, 8, and 12 days of adulthood. An agar plug soaked with 1 μL of the repellent 2-nonanone (1:10 diluted in ethanol) was attached to the plate lid and worm movement was recorded 2 minutes thereafter for 1 minute. Analysis using the MAT showed constant aging-associated decline in mean speed. b Speed distributions within worm populations were unimodal, indicating that the mean value was not biased by a subpopulation of paralyzed or dead worms. The number of speed points in both (a) and (b) that we averaged on were 30,512 for Day 1; 23,672 for Day 4; 9401 for Day 8; and 6240 for day 12. c CF512 worms were grown to days 1, 6, and 12 of adulthood on either control bacteria (EV) or on daf-2 RNAi bacteria. Repellent was added and worm movement was analyzed as in (a). Analysis using the MAT confirmed that reduced insulin/IGF-1 signaling (IIS) slows age-associated motility decline. d AM140 worms expressing the aggregative polyQ35-YFP fusion protein were grown to days 2, 5, and 8 of adulthood on either control EV or daf-2 RNAi bacteria, and speed was scored as in (a). Note that x-axis is drawn to scale with panel c. Speed declines as worms age and proteotoxicity progresses, but reduced IIS slows motility decline. Error bars denote SEM of three biological repeats for panel a and four biological repeats for panels c and d. Each biological repeat consists of 2–3 replicate plates with dozens of worms per plate. Detailed data points are provided in Additional file 13: Table S1.* P < 0.05; ** P < 0.005; *** P < 0.0005
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Fig7: The Multi-Animal Tracker (MAT) is compatible with accurate high-throughput measurements of aging- and neurodegenerative-associated locomotion decline. a Synchronized CF512 worms were grown to 1, 4, 8, and 12 days of adulthood. An agar plug soaked with 1 μL of the repellent 2-nonanone (1:10 diluted in ethanol) was attached to the plate lid and worm movement was recorded 2 minutes thereafter for 1 minute. Analysis using the MAT showed constant aging-associated decline in mean speed. b Speed distributions within worm populations were unimodal, indicating that the mean value was not biased by a subpopulation of paralyzed or dead worms. The number of speed points in both (a) and (b) that we averaged on were 30,512 for Day 1; 23,672 for Day 4; 9401 for Day 8; and 6240 for day 12. c CF512 worms were grown to days 1, 6, and 12 of adulthood on either control bacteria (EV) or on daf-2 RNAi bacteria. Repellent was added and worm movement was analyzed as in (a). Analysis using the MAT confirmed that reduced insulin/IGF-1 signaling (IIS) slows age-associated motility decline. d AM140 worms expressing the aggregative polyQ35-YFP fusion protein were grown to days 2, 5, and 8 of adulthood on either control EV or daf-2 RNAi bacteria, and speed was scored as in (a). Note that x-axis is drawn to scale with panel c. Speed declines as worms age and proteotoxicity progresses, but reduced IIS slows motility decline. Error bars denote SEM of three biological repeats for panel a and four biological repeats for panels c and d. Each biological repeat consists of 2–3 replicate plates with dozens of worms per plate. Detailed data points are provided in Additional file 13: Table S1.* P < 0.05; ** P < 0.005; *** P < 0.0005

Mentions: To quantify locomotion during aging, we used synchronized worm populations and measured animal motility as they age. As expected, we found a steady and significant decline in animal motility that positively correlated with age (Fig. 7a, b). Moreover, as velocity is considerably low in aged animals, our tracker faithfully identifies multiple worms at a time to score minimal displacement events at the single pixels per second resolution. Importantly, the significant decline in the average animal speed was not due to possible death of a fraction of the animals (for which we may score a zero speed). To rule out this possibility, we set our tracking algorithm to discard worm events that showed no activity throughout the experiment. This ensured that dead animals would not skew and bias the experimental results.Fig. 7


A multi-animal tracker for studying complex behaviors
The Multi-Animal Tracker (MAT) is compatible with accurate high-throughput measurements of aging- and neurodegenerative-associated locomotion decline. a Synchronized CF512 worms were grown to 1, 4, 8, and 12 days of adulthood. An agar plug soaked with 1 μL of the repellent 2-nonanone (1:10 diluted in ethanol) was attached to the plate lid and worm movement was recorded 2 minutes thereafter for 1 minute. Analysis using the MAT showed constant aging-associated decline in mean speed. b Speed distributions within worm populations were unimodal, indicating that the mean value was not biased by a subpopulation of paralyzed or dead worms. The number of speed points in both (a) and (b) that we averaged on were 30,512 for Day 1; 23,672 for Day 4; 9401 for Day 8; and 6240 for day 12. c CF512 worms were grown to days 1, 6, and 12 of adulthood on either control bacteria (EV) or on daf-2 RNAi bacteria. Repellent was added and worm movement was analyzed as in (a). Analysis using the MAT confirmed that reduced insulin/IGF-1 signaling (IIS) slows age-associated motility decline. d AM140 worms expressing the aggregative polyQ35-YFP fusion protein were grown to days 2, 5, and 8 of adulthood on either control EV or daf-2 RNAi bacteria, and speed was scored as in (a). Note that x-axis is drawn to scale with panel c. Speed declines as worms age and proteotoxicity progresses, but reduced IIS slows motility decline. Error bars denote SEM of three biological repeats for panel a and four biological repeats for panels c and d. Each biological repeat consists of 2–3 replicate plates with dozens of worms per plate. Detailed data points are provided in Additional file 13: Table S1.* P < 0.05; ** P < 0.005; *** P < 0.0005
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Fig7: The Multi-Animal Tracker (MAT) is compatible with accurate high-throughput measurements of aging- and neurodegenerative-associated locomotion decline. a Synchronized CF512 worms were grown to 1, 4, 8, and 12 days of adulthood. An agar plug soaked with 1 μL of the repellent 2-nonanone (1:10 diluted in ethanol) was attached to the plate lid and worm movement was recorded 2 minutes thereafter for 1 minute. Analysis using the MAT showed constant aging-associated decline in mean speed. b Speed distributions within worm populations were unimodal, indicating that the mean value was not biased by a subpopulation of paralyzed or dead worms. The number of speed points in both (a) and (b) that we averaged on were 30,512 for Day 1; 23,672 for Day 4; 9401 for Day 8; and 6240 for day 12. c CF512 worms were grown to days 1, 6, and 12 of adulthood on either control bacteria (EV) or on daf-2 RNAi bacteria. Repellent was added and worm movement was analyzed as in (a). Analysis using the MAT confirmed that reduced insulin/IGF-1 signaling (IIS) slows age-associated motility decline. d AM140 worms expressing the aggregative polyQ35-YFP fusion protein were grown to days 2, 5, and 8 of adulthood on either control EV or daf-2 RNAi bacteria, and speed was scored as in (a). Note that x-axis is drawn to scale with panel c. Speed declines as worms age and proteotoxicity progresses, but reduced IIS slows motility decline. Error bars denote SEM of three biological repeats for panel a and four biological repeats for panels c and d. Each biological repeat consists of 2–3 replicate plates with dozens of worms per plate. Detailed data points are provided in Additional file 13: Table S1.* P < 0.05; ** P < 0.005; *** P < 0.0005
Mentions: To quantify locomotion during aging, we used synchronized worm populations and measured animal motility as they age. As expected, we found a steady and significant decline in animal motility that positively correlated with age (Fig. 7a, b). Moreover, as velocity is considerably low in aged animals, our tracker faithfully identifies multiple worms at a time to score minimal displacement events at the single pixels per second resolution. Importantly, the significant decline in the average animal speed was not due to possible death of a fraction of the animals (for which we may score a zero speed). To rule out this possibility, we set our tracking algorithm to discard worm events that showed no activity throughout the experiment. This ensured that dead animals would not skew and bias the experimental results.Fig. 7

View Article: PubMed Central - PubMed

ABSTRACT

Background: Animals exhibit astonishingly complex behaviors. Studying the subtle features of these behaviors requires quantitative, high-throughput, and accurate systems that can cope with the often rich perplexing data.

Results: Here, we present a Multi-Animal Tracker (MAT) that provides a user-friendly, end-to-end solution for imaging, tracking, and analyzing complex behaviors of multiple animals simultaneously. At the core of the tracker is a machine learning algorithm that provides immense flexibility to image various animals (e.g., worms, flies, zebrafish, etc.) under different experimental setups and conditions. Focusing on C. elegans worms, we demonstrate the vast advantages of using this MAT in studying complex behaviors. Beginning with chemotaxis, we show that approximately 100 animals can be tracked simultaneously, providing rich behavioral data. Interestingly, we reveal that worms&rsquo; directional changes are biased, rather than random &ndash; a strategy that significantly enhances chemotaxis performance. Next, we show that worms can integrate environmental information and that directional changes mediate the enhanced chemotaxis towards richer environments. Finally, offering high-throughput and accurate tracking, we show that the system is highly suitable for longitudinal studies of aging- and proteotoxicity-associated locomotion deficits, enabling large-scale drug and genetic screens.

Conclusions: Together, our tracker provides a powerful and simple system to study complex behaviors in a quantitative, high-throughput, and accurate manner.

Electronic supplementary material: The online version of this article (doi:10.1186/s12915-017-0363-9) contains supplementary material, which is available to authorized users.

No MeSH data available.